Biodegradable nanoparticulate or microparticulate systems (polymeric particulate systems: PPS) constructed with biocompatible, biodegradable and non-toxic polymers have been used frequently as controlled release delivery vehicles in many industries [1-6]. There is a wide range of polymers that have been applied to obtain nanosystems, microsystems, films or implants, for example polyesters, polyanhydrides, proteins and polysaccharides, etc. [7-14].
One of the most relevant aspects related to polymeric particulate systems (PPS) production is the complexity of industrial scale up processes, which has been considered an important and critical factor for the commercialization of these systems. Many techniques have been developed to prepare PPS for the delivery of drugs such as emulsification or solvent evaporation techniques which involve the use of organic toxic volatile solvents (e.g., dichloromethane, ethyl acetate, chloroform, acetone, ethanol etc.), and applying special complex devices such as high shear homogenizers, supercritical fluid technology or spray dryers. The implementation of said techniques at large-scale production is still a challenge, as it requires defined steps which include process feasibility, formulation optimization, process optimization, scale-up and validation in order to develop quality products and provide a rational approach for production steps including drug concentration and polymer concentration, processing operations, particle size, drug stability or entrapment efficiency. In addition, to develop edible delivery systems suitable for food applications, regulations require that solvents and ingredients are either generally recognized as safe or listed by the Food and Drug Administration as processing aids.
Zein is a plant protein isolated from corn or maize belonging to a family of prolamines which are composed of high amounts of hydrophobic amino acids, such as proline, glutamine and asparagine. Zein is clear, odorless, non-toxic, biodegradable and water-insoluble vegetable protein. Zein has been investigated and used as a polymer in the pharmaceutical, medical, food, cosmetic, adhesive and packaging industries [15].
In the food and pharmaceutical industries, zein has been used, for example, to film-coat materials [16-17] and to form PPS for drug delivery strategies such as nanoparticles [18-20], microparticles [21-26] or polymer-based sustained-release implant drug delivery [27]. Various methods have been proposed to obtain zein particles. The majority of the methods applied to prepare particulate systems are based on the use of volatile organic solvents-aqueous solutions mixture such as ethanol or acetone-water systems to dissolve zein. Then, zein solution is added to other miscible solvent to precipitate zein (solvent displacement-evaporation method) forming colloidal particles. In this context, nanoparticles were prepared by the addition of a stock solution of zein dissolved in 85% ethanol to deionized water [28]. In a similar way, 5-fluorouracil-loaded zein nanoparticles for liver targeting were prepared by dissolving both 5-fluorouracil and zein in 70% ethanol (w/w) with the help of ultrasonic force. The resulting solution was immediately added to distilled water [29]. In a similar way, volatile oil containing nanocapsules were prepared from zein solution in 85% ethanol dispersed with high-speed mixing into 40 mL of water containing 0.01% of silicone fluid [20]. A supercritical anti-solvent process was applied to synthesize micro- and nanoparticles of zein for edible delivery systems of bioactive compounds [30].
U.S. Pat. No. 5,324,351 discloses a solvent mixture to dissolve zein comprising water and from about 60 to about 90 percent of a volatile organic solvent selected from the group consisting of ethanol, acetone and mixtures thereof. Then zein is precipitated by pouring said solution of zein as a stream into an aqueous phase under continuous mixing such that the zein precipitates as fine particles in a colloidal dispersion and finally the organic solvent is removed to obtain an aqueous dispersion comprising from about 0.1 to about 10 percent w/v of zein [31].
U.S. Patent Application 2011/0091565 discloses a method of producing non-immunogenic nanoparticles comprising a hydrophobic water-insoluble protein such as zein by dissolving said protein in a hydroalcoholic solvent (a mixture of ethanol and water) to provide a first aqueous phase solution. Then, a buffering agent is added to the first aqueous phase solution in the presence of a surfactant and a phospholipid to produce a second aqueous phase solution having a pH of between approximately pH 6.8 and approximately pH 7.4. After that, said second aqueous phase solution is processed to effect a reduction in diameter size of particles within the solution by ultrasonic shear, high pressure homogenization or a combination thereof, and finally the residual solvent is evaporated to produce nanoparticles having a diameter size of less than approximately 400 nm. Said method also requires the application of constant ultrasonic energy and a evaporation technique to eliminate the volatile solvent ethanol.
In all cases, the main drawback of the aforementioned methods to obtain zein nanoparticles is the need to apply some techniques to eliminate the volatile organic solvent, such as evaporation under reduced pressure, lyophilization or spray drying.
It is therefore necessary to develop a simple in situ self-assembly method that allows the spontaneous formation of nanoparticles constructed with biocompatible and biodegradable vegetable hydrophobic proteins, particularly zein, said method avoiding the use of volatile organic solvents or complex techniques such as high shear homogenization, supercritical fluid technology or spray dryers and allowing the effective encapsulation of molecules in cationic or anionic nanoparticles at large scale.